M6a-coupled effector protein expression system and methods of making and using same

Abstract

The present disclosure provides systems and methods for m6A-dependent delivery and m6A-dependent delivery targeted of a polypeptide to a cell. In certain embodiments, compositions, systems, and methods are provided that provide for m6A-dependent delivery of effector proteins, for example, effector proteins, such as a tumor suppression proteins and m6a regulation systems, mediated by CRISPRi in embodiments.

What is claimed is: 
     
         1 . An expression system comprising:
 (a) a first DNA construct comprising a polynucleotide encoding a fusion protein, wherein the fusion protein comprises an N 6 -methyladenosine (m 6 A) binding domain of a YT521-B homology (YTH) domain-containing protein fused to a catalytic domain of a cytidine deaminase or a catalytic domain of an adenosine deaminase; and   (b) a second DNA construct comprising a polynucleotide encoding a heterologous polypeptide, the polynucleotide encoding a heterologous polypeptide comprising:
 i. a polynucleotide encoding an effector protein; 
 ii. a polynucleotide encoding a m 6 A sensor sequence; and 
 iii. a polynucleotide encoding a dihydrofolate reductase (DHFR). 
   
     
     
         2 . The expression system of  claim 1 , wherein the m 6 A binding domain comprises a sequence having at least 90% or greater sequence identity to SEQ ID NOs: 66 or 108-116. 
     
     
         3 . The expression system of  claim 1 , wherein the m 6 A binding domain is fused to the catalytic domain via a peptide linker. 
     
     
         4 . The expression system of  claim 1 , wherein the catalytic domain comprises a polypeptide having at least 95% identity to SEQ ID NO 78 or a catalytic fragment thereof, SEQ ID NO: 79 or a catalytic fragment thereof; SEQ ID NO: 80 or a catalytic fragment thereof; or SEQ ID NO: 81. 
     
     
         5 . The expression system of  claim 1 , wherein a vector comprises the first DNA construct. 
     
     
         6 . The expression system of  claim 1 , wherein a vector comprises the second DNA construct. 
     
     
         7 . The expression system of  claim 1 , wherein a vector comprises the first DNA construct and the second DNA construct. 
     
     
         8 . The expression system of  claim 1 , wherein the nucleic acid sequence encoding a fusion protein, the nucleic acid sequence encoding a heterologous polypeptide and a polypeptide encoding dihydrofolate reductase (DHFR), or both, are operably linked to a first promoter. 
     
     
         9 . The expression system of claim  9 , wherein the system further comprises a nucleic acid sequence encoding a selectable marker operably linked to a second promoter. 
     
     
         10 . The expression system of  claim 9 , wherein the first promoter is a constitutive or an inducible promoter. 
     
     
         11 . The expression system of  claim 1 , wherein the cytidine deaminase is APOBEC-1. 
     
     
         12 . The expression system of  claim 1 , wherein the effector protein is a tumor suppressor protein or a catalytically dead RNA-guided endonuclease. 
     
     
         13 . The expression system of claim  13 , wherein the tumor suppressor protein is suppressor of cytokine signaling 2 (SOC2) or p53 or one of the proteins listed in Table 1. 
     
     
         14 . The expression system of  claim 13 , wherein the catalytically dead RNA-guided endonuclease is a dCas9 or a dCas13. 
     
     
         15 . A polynucleotide comprising a nucleic acid sequence encoding an effector protein polypeptide, a m 6 A sensor sequence, and a polypeptide encoding dihydrofolate reductase (DHFR). 
     
     
         16 . A vector comprising the expression system of  claim 1 . 
     
     
         17 . A host cell comprising the expression system of  claim 1  or the vector of claim  17 . 
     
     
         18 . A non-human transgenic animal comprising the host cell of claim  18 . 
     
     
         19 . A kit comprising the expression system of  claim 1 . 
     
     
         20 . A method of increasing expression of a tumor suppressor protein in one or more cells, comprising introducing the expression system of  claim 1  into the one or more cells. 
     
     
         21 . The method of claim  21 , wherein the one or more cells are hepatocellular carcinoma (HCC) cells. 
     
     
         22 . (canceled) 
     
     
         23 . A method of reducing M6A effector regulator expression, comprising:
 introducing an expression system into a subject having or suspected of having a cancer, wherein the expression system comprises:   (a) a first DNA construct comprising a polynucleotide encoding a fusion protein, wherein the fusion protein comprises an N 6 -methyladenosine (m 6 A) binding domain of a YT521-B homology (YTH) domain-containing protein fused to a catalytic domain of a cytidine deaminase or a catalytic domain of an adenosine deaminase; and   (b) a second DNA construct comprising a polynucleotide encoding a heterologous polypeptide, the polynucleotide encoding a heterologous polypeptide comprising:
 iv. a polynucleotide encoding a catalytically-dead RNA-guided endonuclease; 
 v. a polynucleotide encoding a m 6 A sensor sequence; and 
 vi. a polynucleotide encoding a dihydrofolate reductase (DHFR); 
   (c) an sgRNA configured to bind to an m6a regulator.   
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . (canceled) 
     
     
         28 . A method of reducing M6A hypermethylation, comprising:
 introducing an expression system into a subject having or suspected of having a cancer, wherein the expression system comprises:   (a) a first DNA construct comprising a polynucleotide encoding a fusion protein, wherein the fusion protein comprises an N 6 -methyladenosine (m 6 A) binding domain of a YT521-B homology (YTH) domain-containing protein fused to a catalytic domain of a cytidine deaminase or a catalytic domain of an adenosine deaminase; and   (b) a second DNA construct comprising a polynucleotide encoding a heterologous polypeptide, the polynucleotide encoding a heterologous polypeptide comprising:
 vii. a polynucleotide encoding a catalytically-dead RNA-guided endonuclease; 
 viii. a polynucleotide encoding a m 6 A sensor sequence; and 
 ix. a polynucleotide encoding a dihydrofolate reductase (DHFR); 
   (c) an sgRNA configured to bind to an m6a regulator.   
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . (canceled) 
     
     
         31 . (canceled) 
     
     
         32 . A method of inhibiting a cancer cell, the method comprising:
 introducing the expression system of  claim 1  into the cancer cell, wherein the cancer cell comprises m 6 A RNA hypermethylation, and wherein the second DNA construct comprising a polynucleotide encoding an effector protein, the effector protein comprising a tumor suppressor protein.   
     
     
         33 . (canceled) 
     
     
         34 . (canceled) 
     
     
         35 . (canceled) 
     
     
         36 . (canceled) 
     
     
         37 . (canceled) 
     
     
         38 . (canceled) 
     
     
         39 . (canceled) 
     
     
         40 . (canceled) 
     
     
         41 . (canceled) 
     
     
         42 . (canceled) 
     
     
         43 . A method of treating a subject having a cancer characterized by m 6 A RNA hypermethylation, the method comprising inhibiting a cancer cell according to the method of  claim 32 . 
     
     
         44 . The method of  claim 43 , wherein the cancer comprises at least one of acute myeloid leukemia (AML), glioblastoma (GBM), lung cancer, endometrial cancer, cervical cancer, ovarian cancer, breast cancer, colorectal cancer (CRC), a hepatocellular carcinoma (HCC), pancreatic cancer, gastric cancer, prostate cancer, or renal cell carcinoma. 
     
     
         45 . (canceled) 
     
     
         46 . (canceled) 
     
     
         47 . (canceled)